Application of a sturgeon roe peptide in improving premature ovarian failure and brain health

Abstract

This invention discloses the application of sturgeon roe peptides in improving premature ovarian failure and brain health, belonging to the field of bioactive peptide technology. The sturgeon roe peptides prepared by this invention have been experimentally verified to improve premature ovarian failure by improving hormone levels and enhancing antioxidant and anti-inflammatory capabilities, while also possessing brain health-improving effects. They can be widely used in common foods and medicines.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and more specifically to the application of sturgeon roe peptide in improving premature ovarian failure and brain health. Background Technology

[0002] Premature ovarian failure (POF) is a reproductive system disease affecting 1%-5% of women under 40 years old worldwide. Its core pathological feature is the premature decline of ovarian reserve, which leads to a sharp drop in estrogen levels and abnormally high levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), resulting in serious complications such as infertility, amenorrhea, osteoporosis, and cardiovascular disease.

[0003] Meanwhile, premature ovarian failure not only leads to reproductive system disorders, but can also trigger neurodegenerative diseases through estrogen deficiency. Estrogen plays a crucial role in protecting neurons in brain regions such as the hippocampus and prefrontal cortex. A decline in estrogen levels accelerates β-amyloid protein deposition, oxidative stress damage, and loss of synaptic plasticity, ultimately manifesting as cognitive decline, mood disorders, and an increased risk of Alzheimer's disease.

[0004] Currently, traditional treatments for premature ovarian failure have significant limitations. For example, hormone replacement therapy (HRT): long-term use of exogenous estrogen may increase the risk of breast cancer, endometrial cancer, and thrombosis, and it cannot reverse ovarian tissue atrophy. Immunotherapy: immunosuppressant therapy targeting autoimmune ovarian damage has serious adverse reactions, and its long-term efficacy is unstable. Stem cell therapy: although animal studies show that mesenchymal stem cells can improve ovarian function, its clinical translation faces problems such as insufficient safety assessment, ethical controversies, and high costs.

[0005] Therefore, how to develop a dual-target biological agent that combines ovarian repair and neuroprotection is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an application of sturgeon caviar peptide in improving premature ovarian failure and brain health, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of a sturgeon roe peptide in the preparation of products that improve premature ovarian failure and brain health.

[0009] Furthermore, the preparation method of the above-mentioned sturgeon roe peptide (lyophilized powder) specifically includes the following steps:

[0010] (1) Thaw the sturgeon roe, add water, stir evenly, grind and crush, adjust the pH value to obtain sturgeon roe solution;

[0011] (2) Heat the sturgeon roe solution and then let it cool naturally;

[0012] (3) First, add alkaline protease to the cooled sturgeon roe solution, stir evenly for enzymatic hydrolysis, and then add sodium octenyl succinate starch and vitamin E powder.

[0013] (4) The enzyme was inactivated, cooled naturally, and the pH value was adjusted to obtain sturgeon roe hydrolysate;

[0014] (5) The sturgeon roe enzymatic hydrolysate was coarsely filtered and centrifuged. The supernatant was then adsorbed through an activated carbon tandem column and filtered. The filtrate was then collected to obtain the sturgeon roe peptide solution.

[0015] (6) Plate and frame filtration, take the filtrate, freeze dry under vacuum, sieve, and package to obtain sturgeon roe peptide (freeze-dried powder).

[0016] Furthermore, in step (1) above, the mass ratio of sturgeon roe to water is 1:6.

[0017] Furthermore, in step (1) above, the grinding equipment is a colloid mill, and the grinding is performed once; the specific operation of adjusting the pH value is as follows: add a saturated NaOH solution, stir evenly, and adjust the pH value of the material to 8.

[0018] Furthermore, in step (2) above, the temperature is raised to 80°C and held for 10 minutes; then naturally cooled to 55°C.

[0019] Furthermore, in step (3) above, the amount of alkaline protease added is 1% of the protein content of sturgeon roe.

[0020] Furthermore, in step (3) above, the enzymatic hydrolysis temperature is 55℃ and the time is 8h;

[0021] Furthermore, in step (3) above, the amount of sodium octenyl succinate starch added is 10 g / L; the vitamin E content in the vitamin E powder is 50%, and the amount added is 12 mg / L;

[0022] The further beneficial effect of the above-mentioned method is that the encapsulating agent is a key component in the preparation of sturgeon roe peptide (lyophilized powder). By selecting food-grade encapsulating agent raw materials with good film-forming properties, water resistance, and stability, the present invention can effectively encapsulate sturgeon roe peptide.

[0023] Furthermore, in step (4) above, the enzyme inactivation temperature is 90-100℃ and the time is 10-15min; it is naturally cooled to room temperature; the specific operation of adjusting the pH value is as follows: add hydrochloric acid solution, stir evenly, and adjust the pH value of the material to 7.

[0024] Furthermore, in step (5) above, the tools used for coarse filtration are filter bags and 200-mesh screens; the centrifugal speed is 5000-20000 r / min; and the activated carbon series column adsorption is performed 3 times.

[0025] Furthermore, in step (6) above, the filter paper precision of the plate and frame filter is 0.1 μm, and the number of filtrations is 3; the vacuum degree of vacuum freeze drying is 100 Pa, the temperature is -45℃, and the time is 2000 min; the sieve mesh number of the sieve is 80 mesh; and the packaging material is a PE bag.

[0026] The further beneficial effect of the above-mentioned method is that mixing and homogenizing the sturgeon roe peptide solution with the encapsulating agent solution in a certain proportion is an important step in the preparation of sturgeon roe peptide (lyophilized powder). By selecting appropriate enzyme preparations, temperature and time and other process parameters, the present invention ensures the effectiveness of sturgeon roe peptide.

[0027] Vacuum freeze-drying is a key step in the preparation of sturgeon roe peptides (freeze-dried powder). This invention ensures the quality and stability of sturgeon roe peptides by selecting appropriate process parameters such as vacuum degree, temperature and time.

[0028] Post-processing of the obtained sturgeon roe peptides (freeze-dried powder), such as sieving and packaging, is an important step in ensuring product quality and stability. This invention ensures that the product meets relevant standards by selecting appropriate sieve mesh size and packaging materials and other process parameters.

[0029] Furthermore, the above products include liquids, ointments, capsules, tablets, powders, and pills.

[0030] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The preparation method of the present invention has the advantages of simple operation, low cost and high encapsulation yield, which is of great significance for improving the stability and bioactivity of sturgeon roe peptides.

[0032] 2. The sturgeon roe peptide prepared by this invention has the effect of improving premature ovarian failure and brain health, and can be widely used in ordinary liquids, ointments, capsules, tablets, powders and pills. Attached Figure Description

[0033] Figure 1 Vaginal smears from the estrous cycle of normal mice;

[0034] Figure 2 Vaginal smears of mice in the model group during their estrous cycle;

[0035] Figure 3 Vaginal smears of mice in the sturgeon roe peptide group during their estrous cycle;

[0036] Figure 4 Images of ovarian tissue sections from each treatment group;

[0037] Figure 5 Uterine tissue from each treatment group;

[0038] Figure 6 TUNEL staining results for each treatment group. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] The preparation method of sturgeon roe peptide (lyophilized powder) specifically includes the following steps:

[0042] (1) Thaw sturgeon roe, take 1 kg and add 6 kg of deionized water, stir evenly, grind once with a colloid mill to break it up, add saturated NaOH solution, stir evenly, adjust the pH value of the material to 8, and obtain sturgeon roe solution.

[0043] (2) Heat the sturgeon roe solution to 80°C and hold for 10 minutes, then let it cool naturally to 55°C;

[0044] (3) First, add 1% alkaline protease of sturgeon roe protein to the cooled sturgeon roe solution, stir evenly, and enzymatically hydrolyze at 55℃ for 8 hours. Then, add 10g sodium octenyl succinate starch and 12mg vitamin E powder with a content of 50% to each L of sturgeon roe solution.

[0045] (4) Inactivate the enzyme at 90℃ for 15 min, cool naturally to room temperature, add hydrochloric acid solution, stir evenly, adjust the pH of the material to 7, and obtain sturgeon roe hydrolysate;

[0046] (5) First, the sturgeon roe enzymatic hydrolysate is coarsely filtered through a filter bag and a 200-mesh sieve, then centrifuged at 5000 r / min, and the supernatant is taken. It is then adsorbed three times through a series of activated carbon columns, filtered, and the filtrate is taken to obtain sturgeon roe peptide solution.

[0047] (6) First, filter the sturgeon roe peptide solution through a plate and frame filter paper with a filter paper precision of 0.1 μm three times, take the filtrate and sterilize it at high temperature; then freeze-dry it under vacuum conditions of 100 Pa and -45 °C for 2000 min, sieve it through an 80 mesh sieve, and finally package it in a PE bag to obtain sturgeon roe peptide (freeze-dried powder).

[0048] Example 2

[0049] The preparation method of sturgeon roe peptide (lyophilized powder) specifically includes the following steps:

[0050] (1) Thaw sturgeon roe, take 1 kg and add 6 kg of deionized water, stir evenly, grind once with a colloid mill to break it up, add saturated NaOH solution, stir evenly, adjust the pH value of the material to 8, and obtain sturgeon roe solution.

[0051] (2) Heat the sturgeon roe solution to 80°C and hold for 10 minutes, then let it cool naturally to 55°C;

[0052] (3) First, add 1% alkaline protease of sturgeon roe protein to the cooled sturgeon roe solution, stir evenly, and enzymatically hydrolyze at 55℃ for 8 hours. Then, add 10g sodium octenyl succinate starch and 12mg vitamin E powder with a content of 50% to each L of sturgeon roe solution.

[0053] (4) Inactivate the enzyme at 95℃ for 12 min, cool naturally to room temperature, add hydrochloric acid solution, stir evenly, adjust the pH of the material to 7, and obtain sturgeon roe hydrolysate;

[0054] (5) First, the sturgeon roe enzymatic hydrolysate is coarsely filtered through a filter bag and a 200-mesh sieve, then centrifuged at 10,000 r / min, and the supernatant is taken. It is then adsorbed three times through a series of activated carbon columns, filtered, and the filtrate is taken to obtain sturgeon roe peptide solution.

[0055] (6) First, filter the sturgeon roe peptide solution through a plate and frame filter paper with a filter paper precision of 0.1 μm three times, take the filtrate and sterilize it at high temperature; then freeze-dry it under vacuum conditions of 100 Pa and -45 °C for 2000 min, sieve it through an 80 mesh sieve, and finally package it in a PE bag to obtain sturgeon roe peptide (freeze-dried powder).

[0056] Example 3

[0057] The preparation method of sturgeon roe peptide (lyophilized powder) specifically includes the following steps:

[0058] (1) Thaw sturgeon roe, take 1 kg and add 6 kg of deionized water, stir evenly, grind once with a colloid mill to break it up, add saturated NaOH solution, stir evenly, adjust the pH value of the material to 8, and obtain sturgeon roe solution.

[0059] (2) Heat the sturgeon roe solution to 80°C and hold for 10 minutes, then let it cool naturally to 55°C;

[0060] (3) First, add 1% alkaline protease of sturgeon roe protein to the cooled sturgeon roe solution, stir evenly, and enzymatically hydrolyze at 55℃ for 8 hours. Then, add 10g sodium octenyl succinate starch and 12mg vitamin E powder with a content of 50% to each L of sturgeon roe solution.

[0061] (4) Inactivate the enzyme at 100℃ for 10 min, cool naturally to room temperature, add hydrochloric acid solution, stir evenly, adjust the pH of the material to 7, and obtain sturgeon roe hydrolysate;

[0062] (5) First, the sturgeon roe enzymatic hydrolysate is coarsely filtered through a filter bag and a 200-mesh sieve, then centrifuged at 20,000 r / min, and the supernatant is taken. It is then adsorbed three times through a series of activated carbon columns, filtered, and the filtrate is taken to obtain the sturgeon roe peptide solution.

[0063] (6) First, filter the sturgeon roe peptide solution through a plate and frame filter paper with a filter paper precision of 0.1 μm three times, take the filtrate and sterilize it at high temperature; then freeze-dry it under vacuum conditions of 100 Pa and -45 °C for 2000 min, sieve it through an 80 mesh sieve, and finally package it in a PE bag to obtain sturgeon roe peptide (freeze-dried powder).

[0064] Performance testing

[0065] Research on the effect of fish roe peptides on improving premature ovarian failure

[0066] Based on a mouse model of premature ovarian failure and the hypothalamic-pituitary-ovarian axis, this study investigated the ameliorative effect of sturgeon caviar peptides on premature ovarian failure in female mice by measuring indicators such as estrous cycle, ovarian cell apoptosis, serum hormone levels, expression of genes related to hormone synthesis in the ovary and hypothalamus, histopathology, and serum metabolomics.

[0067] 1. Animal experimental research methods

[0068] 1.1 Experimental Grouping

[0069] Balb / C female mice (8-10 weeks old, 27 in total) were randomly divided into 3 groups: control group, premature ovarian failure model group (model group), and sturgeon caviar peptide administration experimental group (experimental group), with 9 mice in each group. Each group was weighed and marked, and then housed in separate cages.

[0070] 1.2 Experimental Methods and Results

[0071] Mice in the model and experimental groups were intraperitoneally injected with 100 mg / kg cyclophosphamide for two consecutive weeks, while the control group was injected intraperitoneally with the same volume of physiological saline. For six consecutive weeks, mice were administered the sturgeon roe peptide group via gavage once daily. The sturgeon roe peptide group was administered 115 mg / kg of the sturgeon roe peptide (lyophilized powder) prepared in Example 2 via gavage, while the model and control groups were administered the same volume of physiological saline via gavage. For the following 20 days, the blank control and model groups were given a 0.5% sodium carboxymethyl cellulose aqueous solution (solvent control).

[0072] 1.3 Measurement Indicators

[0073] (1) Vaginal exfoliative cytology and estrous cycle assessment

[0074] For 10 consecutive days starting from model establishment and for another 10 consecutive days starting from week 4 of sample intervention, vaginal exfoliated cell smears were examined daily at 10:00 AM to observe the estrous cycle of the mice. Using a 20 μL pipette, 10 μL of physiological saline was drawn and slowly inserted into the mouse vagina approximately 0.5 cm deep. The pipette was blown 2-3 times to aspirate the saline, which was then evenly spread onto a clean glass slide, allowed to dry, and stained with H&E.

[0075] (2) Ovarian coefficient

[0076] After modeling and intervention, blood was collected from the orbital cavity, the mice were euthanized by dislocation of the neck, the abdomen was dissected, and both ovaries were removed and weighed. The organ index of both ovaries was calculated according to the formula: weight of both ovaries (mg) ÷ weight of mouse (g) × 100%.

[0077] (3) Serum hormone levels

[0078] Blood was collected from the ocular venous plexus after the 6th week of intervention. After standing at room temperature for 2 hours, the blood was centrifuged at 4°C for 15 minutes (4000 rpm), and the supernatant was stored at -80°C for later analysis. Serum levels of testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), cyclic adenosine monophosphate (cAMP), and gonadotropin-releasing hormone (GnRH) were measured using an ELISA kit.

[0079] (4) Brain indicators

[0080] After the intervention ended in week 6, the mice were dissected, and the hippocampus and prefrontal cortex were harvested. The levels of brain-derived neurotrophic factor (BDNF), acetylcholinesterase (AChE), and acetylcholine (ACh) in the two sites were measured using an ELISA kit.

[0081] (5) Pathological tissue observation

[0082] After the sixth week of the experiment, mice were dissected, and ovarian and uterine tissues were fixed in 4% paraformaldehyde. Following routine dehydration, clearing, paraffin embedding, sectioning, and dewaxing, sections were stained with Mayer's hematoxylin for 5–7 minutes, washed with pure water, and then blued back to blue. After air drying, the sections were mounted with neutral resin, and finally examined under a microscope to observe changes in the morphology of the ovarian and uterine tissues.

[0083] (6) Tunel fluorescence method

[0084] The apoptosis level of mouse ovarian cells in each group was determined using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) method. Ovarian sections were fixed with 4% PFA for 1 h, then incubated with 3% H2O2 for 10 min, followed by incubation with 0.1-0.5% Triton X-100 for 5 min. Finally, the cells were nucleated with 40,6-diamidinyl-2-phenylindole (DAPI) for 8 min, and apoptotic cells in the ovary were stained using an in situ cell death detection kit. Finally, TUNEL-positive cells were observed using fluorescence microscopy, and the apoptotic cell positivity rate was calculated using ImageJ software.

[0085] (7) Determination of markers of oxidative stress

[0086] Ovarian tissue samples (0.15–0.2 g) were placed in 1.5 mL centrifuge tubes and homogenized with 0.9% saline solution at a ratio of 1:9 (w / v) to prepare a 10% ovarian tissue homogenate. The homogenate was then centrifuged twice at 13,000 rpm for 10 minutes, discarding the precipitate and retaining the supernatant for subsequent oxidation index determination. Following the instructions of the oxidation index assay kit, the activities of oxidation-related factors MDA, SOD, GSH-Px, and inflammation-related factors IL-6, IL-1β, and TNF-α were measured at 532 nm and 560 nm, respectively.

[0087] 2. Data and Discussion

[0088] 2.1 Ovarian coefficient

[0089] The results of the ovarian coefficient analysis are shown in Table 1. Compared with the control group, the ovarian coefficient of the model group was significantly reduced (P<0.05), indicating that cyclophosphamide treatment did indeed lead to ovarian atrophy and functional decline. The experimental group significantly increased the ovarian coefficient of perimenopausal female mice (P<0.05), and to some extent repaired ovarian function.

[0090] Table 1 Ovarian coefficient

[0091] control group Model group experimental group Ovarian coefficient (%) <![CDATA[0.62 ± 0.07 a ]]> <![CDATA[0.34 ± 0.17 b ]]> <![CDATA[0.5 ± 0.1 a ]]>

[0092] 2.2 Vaginal exfoliative cytology and estrous cycle assessment

[0093] (1) Vaginal exfoliative cytology smear

[0094] Cyclophosphamide treatment can significantly affect the estrous cycle in female mice through its reproductive toxicity, mainly manifesting as cycle irregularities or prolongation. Studies have shown that this drug reduces estrogen levels by inducing apoptosis and oxidative damage in ovarian follicles (especially primordial follicles), interfering with the endocrine regulation of the hypothalamic-pituitary-gonadal axis, thereby leading to irregular estrous cycles or arrest at specific stages (such as the estrus period). Furthermore, high doses or long-term exposure exacerbate this effect, and the degree of damage is dose-dependent, possibly directly related to decreased ovarian reserve and impaired granulosa cell function.

[0095] Mouse estrous cycle as Figure 1-3 As shown, the interestrus phase is characterized by a large number of leukocytes and a small number of epithelial cells. During proestrus, nucleated epithelial cells predominate, with fewer keratinized epithelial cells and leukocytes. During estrus, numerous squamous keratinized epithelial cells are shed, primarily anucleate polygonal cells. During metestrus, nucleated epithelial cells, keratinized epithelial cells, and leukocytes coexist.

[0096] (2) Changes in the estrus cycle

[0097] Estrogenic cycle changes were assessed based on vaginal smears from the estrous cycle. In this experiment, a normal cycle was defined as an estrous cycle of 4-5 days with even distribution of cycles, while an abnormal cycle was defined as a cycle <4 days or >5 days with disordered cycles. As shown in Table 2, the proportion of mice maintaining a normal cycle in the control group was relatively high (66.67%), while the model group was entirely in a disordered cycle (100%). The proportion of normal cycles in the experimental group was 66.67%, improving to the same level as the control group, indicating that sturgeon caviar peptides help repair ovarian function.

[0098] Table 2. Changes in the estrous cycle

[0099] control group Model group experimental group Percentage of normal cycles (%) 66.67 0.00 66.67 Percentage of disordered cycles (%) 33.33 100.00 33.33

[0100] 2.3 Serum hormone levels

[0101] (1) Detection of anti-Müllerian hormone (AMH) content

[0102] AMH is mainly secreted by the granulosa cells of the preantral and small antral follicles of the ovary, and its level directly reflects ovarian reserve function. As shown in Table 3 of this study, the AMH level in the model group was significantly lower than that in the control group (P<0.05), indicating that cyclophosphamide can significantly reduce AMH levels by impairing ovarian function. The AMH level in the experimental group was significantly higher than that in the model group (P<0.05), indicating that the experimental group helps improve ovarian reserve function by increasing AMH levels.

[0103] Table 3. Anti-Müllerian Hormone (AMH) Levels

[0104] control group Model group experimental group AMH (ng / mL) <![CDATA[2570 ± 0 a ]]> <![CDATA[1953 ± 108.5 b ]]> <![CDATA[2370 ± 106.1 a ]]>

[0105] (2) Detection of estradiol E2 content

[0106] Estradiol (E2) is primarily synthesized by ovarian granulosa cells, and its secretion depends on follicular development and the fine-tuned regulation of the hypothalamic-pituitary-ovarian axis (HPO axis). Studies have shown that the reproductive toxicity of cyclophosphamide can induce apoptosis of primordial and antral follicles, directly reducing the number of secretory granulosa cells, while inhibiting the activity of steroid-producing enzymes (such as aromatase Cyp19a1), thus hindering the conversion of testosterone to estradiol. Oxidative stress and mitochondrial dysfunction further exacerbate granulosa cell dysfunction, leading to reduced E2 synthesis. This effect is dose- and time-dependent; high-dose exposure or long-term administration may induce persistently low E2 levels, which are closely associated with estrous cycle disorders, decreased AMH levels, and premature ovarian failure phenotypes. Notably, the decrease in E2 may activate the HPO axis through a negative feedback mechanism, prompting a compensatory increase in FSH, but ovarian failure ultimately leads to overall hormonal axis dysregulation.

[0107] The results are shown in Table 4. Compared with the control group, the E2 content in the cyclophosphamide-treated model group was significantly reduced (P<0.05), indicating ovarian dysfunction. The experimental group significantly increased the serum E2 content in female mice with cyclophosphamide-induced ovarian aging (P<0.05).

[0108] Table 4 Estrogen E2 Levels

[0109] control group Model group experimental group <![CDATA[E2(pmol / mL)]]> <![CDATA[2.28 ± 0.13 a ]]> <![CDATA[1.3 ± 0.1 b ]]> <![CDATA[3.72 ± 0.33 c ]]>

[0110] (3) Detection of follicle-stimulating hormone (FSH) levels

[0111] The decrease in E2 may stimulate negative feedback regulation in female mice, thereby increasing serum FSH levels. As shown in Table 5, compared with the control, the FSH content in the model group was significantly increased, while the FSH level in the serum of female mice in the experimental group was significantly reduced (P<0.05).

[0112] Table 5. Follicle-stimulating hormone (FSH) levels

[0113] control group Model group experimental group FSH (mIU / mL) <![CDATA[6.94 ± 0.44 a ]]> <![CDATA[10.07 ± 1.01 b ]]> <![CDATA[4.81 ± 0.47 c ]]>

[0114] (4) Detection of luteinizing hormone levels

[0115] Cyclophosphamide treatment can lead to abnormally elevated serum luteinizing hormone (LH) levels in female mice. The mechanism is primarily related to impaired ovarian function: cyclophosphamide significantly reduces estradiol (E2) secretion by inducing follicular apoptosis and granulosa cell damage, weakening the negative feedback inhibition of the hypothalamic-pituitary axis and prompting compensatory increases in the secretion of gonadotropin-releasing hormone (GnRH) and pituitary gonadotropins (such as LH and FSH). However, long-term or high-dose exposure may weaken pituitary-ovarian axis regulation due to ovarian failure, ultimately leading to fluctuations or dysregulation of LH levels. This change is often closely related to estrous cycle disorders and overall imbalances in the reproductive endocrine network.

[0116] The results are shown in Table 6. The LH levels in the cyclophosphamide-treated model group were significantly higher than those in the normal group (P<0.05), suggesting an imbalance in the estrous cycle and the overall reproductive endocrine network. Compared to the model group, the experimental group showed a decrease in LH levels.

[0117] Table 6. Luteinizing hormone (LH) levels

[0118] control group Model group experimental group LH (mU / mL) <![CDATA[7.53 ± 0.19 a ]]> <![CDATA[9.50± 0.29 b ]]> <![CDATA[8.68 ± 0.54 c ]]>

[0119] (5) Changes in the FSH / LH ratio

[0120] Cyclophosphamide treatment weakens the promoting effect of FSH on E2 synthesis in granulosa cells by damaging the ovarian microenvironment and interfering with gonadotropin (FSH / LH) signaling. Table 7 shows that the FSH / LH ratio was significantly higher in the model group compared to the control group, indicating ovarian dysfunction. Compared to the model group, the experimental group showed a significant reduction in the FSH / LH ratio and improved ovarian function.

[0121] Table 7 FSH / LH ratio

[0122] control group Model group experimental group FSH / LH <![CDATA[0.92 ± 0.04 a ]]> <![CDATA[1.06 ± 0.16 b ]]> <![CDATA[0.54 ± 0.06 c ]]>

[0123] (6) Testosterone content detection

[0124] Cyclophosphamide treatment may reduce serum testosterone (T) levels in female mice by inhibiting ovarian steroid production. Testosterone is primarily synthesized by theca cells in the ovary and serves as a precursor to estradiol (E2). Its synthesis depends on the activity of steroid-producing enzymes (such as Cyp17a1). Cyclophosphamide-induced theca cell apoptosis, oxidative damage, and mitochondrial dysfunction can disrupt the testosterone synthesis pathway. Low-dose or short-term exposure may lead to fluctuations in T levels, while high-dose or long-term treatment is often accompanied by a significant decrease in T levels, which is associated with reduced E2, follicular arrest, and reproductive endocrine disorders.

[0125] Table 8 shows that the T content in the cyclophosphamide-treated model group was significantly lower than that in the control group (P<0.05), indicating that follicular cells may have problems such as apoptosis, oxidative damage, and mitochondrial disorders, affecting the T synthesis pathway. Compared with the model group, the sample group showed a significant increase in T content (P<0.05).

[0126] Table 8 Testosterone T Levels

[0127] control group Model group experimental group T (ng / mL) <![CDATA[0.28 ± 0.01 a ]]> <![CDATA[0.06 ± 0 c ]]> <![CDATA[0.16 ± 0.01 b ]]>

[0128] (7) Detection of gonadotropin-releasing hormone content

[0129] Cyclophosphamide treatment may indirectly affect the secretion level of gonadotropin-releasing hormone (GnRH) in the serum of female mice by interfering with the negative feedback regulation of the hypothalamus-pituitary-ovarian axis (HPO axis). Under normal circumstances, GnRH is secreted by the hypothalamus and regulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. Cyclophosphamide-induced ovarian damage (such as follicular apoptosis and reduced estradiol (E2) synthesis) significantly reduces the negative feedback inhibition of E2 on the hypothalamus, thereby compensating by activating the pulsatile release of GnRH, prompting the pituitary gland to secrete more LH / FSH to stimulate ovarian function.

[0130] As shown in Table 9, compared with the control group, cyclophosphamide treatment significantly increased the GnRH content in the model group (P<0.05), while the experimental group significantly decreased its content (P<0.05).

[0131] Table 9. Gonadotropin-releasing hormone (GnRH) levels

[0132] control group Model group experimental group GnRH (mIU / mL) <![CDATA[0.63 ± 0.03 ac ]]> <![CDATA[1.33 ± 0.03 b ]]> <![CDATA[1.11 ± 0.05 c ]]>

[0133] 2.5 Brain Indicators

[0134] (1) Analysis of acetylcholine content

[0135] Cyclophosphamide treatment may affect acetylcholine (ACh) levels in the brains of female mice through blood-brain barrier penetration and neurotoxicity. Studies have shown that cyclophosphamide can induce oxidative stress and mitochondrial dysfunction in the central nervous system, inhibit choline acetyltransferase (ChAT) activity, and reduce ACh synthesis; it may also activate acetylcholinesterase (AChE), accelerating ACh degradation. Furthermore, its pro-inflammatory effects (such as increased TNF-α and IL-6) may further impair cholinergic neuronal function. These changes are particularly pronounced in cognitively relevant brain regions such as the hippocampus and cortex, potentially leading to decreased ACh levels and association with chemotherapy-related cognitive impairment (such as impaired learning and memory).

[0136] As shown in Table 10 of this study, compared with the normal group, the ACh levels in the prefrontal cortex and hippocampus of the model group were significantly decreased (P<0.05), suggesting that cyclophosphamide impairs the transmission of memory signals in the brain. In the ACh results of the prefrontal cortex, the experimental group significantly increased the ACh content in both the prefrontal cortex and hippocampus to the control group level (P<0.05).

[0137] Table 10. Acetylcholine (ACh) content in the prefrontal cortex and hippocampus

[0138] control group Model group experimental group ACh in the prefrontal cortex (pg / mL) <![CDATA[970.45±59 a ]]> <![CDATA[750±70.45 b ]]> <![CDATA[929.55±47.2 a ]]> Hippocampal ACh (pg / mL) <![CDATA[1083.33±71.34 a ]]> <![CDATA[817.05±27.32 b ]]> <![CDATA[1069.89±38.09 a ]]>

[0139] (2) Analysis of acetylcholinesterase levels

[0140] Table 11 shows that AChE activity was significantly increased in the prefrontal cortex and hippocampus of the model group (P<0.05), indicating that AChE was overactivated in the model group, which may lead to a large amount of degradation of acetylcholine and further affect the transmission of brain signals. In contrast, the experimental group significantly reduced the increase in AChE activity caused by cyclophosphamide treatment (P<0.05).

[0141] Table 11. Acetylcholinesterase (AChE) levels in the prefrontal cortex and hippocampus

[0142] control group Model group experimental group AChE in the prefrontal cortex (mmol / L) <![CDATA[298.83±8.19 a ]]> <![CDATA[356.8±5.93 b ]]> <![CDATA[292.3±17.01 a ]]> Hippocampal AChE (mmol / L) <![CDATA[267.39±15.29 a ]]> <![CDATA[353.06±8.15 b ]]> <![CDATA[298.49±24.75 a ]]>

[0143] (3) BDNF content analysis

[0144] Cyclophosphamide treatment may significantly reduce the level of brain-derived neurotrophic factor (BDNF) in the brains of female mice through neuroinflammation and oxidative stress pathways. BDNF is a key protein regulating neuronal survival, synaptic plasticity, and cognitive function. Table 12 shows that the BDNF level in the model group was significantly reduced (P<0.05), suggesting that cyclophosphamide treatment may reduce the level of BDNF in the brain through stress, further affecting brain memory. Sturgeon roe peptides can increase the BDNF levels in the prefrontal cortex and hippocampus, but the differences are not significant.

[0145] Table 12. Levels of brain-derived neurotrophic factor (BDNF) in the prefrontal cortex and hippocampus.

[0146] control group Model group experimental group BDNF in the prefrontal cortex (ng / mL) <![CDATA[2011.96±177.62 a ]]> <![CDATA[1465.76±23.83 b ]]> <![CDATA[1656.52±109.14 b ]]> Hippocampal BDNF (ng / mL) <![CDATA[2169.57±163.04 a ]]> <![CDATA[1701.09±52.26 b ]]> <![CDATA[1784.78±60 b ]]>

[0147] 2.6 Pathological tissue observation

[0148] (1) Observation of ovarian tissue

[0149] Cyclophosphamide treatment can significantly disrupt ovarian follicular homeostasis in female mice, manifested as a decrease in mature follicles, an increase in atretic follicles, and depletion of primordial follicle reserves. Specific effects include: ① Decrease in mature follicles: Cyclophosphamide inhibits granulosa cell proliferation, interferes with the FSH / cAMP signaling pathway, and reduces the activity of steroid-producing enzymes (such as Cyp19a1), hindering the development of antral follicles into mature follicles (such as Graff follicles), leading to decreased ovulation function; ② Increase in atretic follicles: The drug induces apoptosis of theca cells and granulosa cells (such as activating the Bax / Caspase-3 pathway), while simultaneously triggering ovarian oxidative stress (such as ROS accumulation and decreased SOD activity), accelerating the atresia process of growing follicles (especially secondary and antral follicles); ③ Depletion of primordial follicle reserves: Cyclophosphamide can directly damage primordial follicles, leading to premature activation or apoptosis of primordial follicles by activating the p53-p21 pathway or disrupting oocyte mitochondrial function. This irreversible damage is a key mechanism of premature ovarian failure and is dose-dependent; high-dose treatment can lead to a sharp reduction in the primordial follicle pool. ④ Associated effects: The above changes are directly related to decreased serum AMH, reduced E2 levels, and estrous cycle disorders; long-term exposure may completely destroy ovarian reproductive endocrine function. Protective strategies (such as antioxidants or follicle protectants) can partially alleviate the follicle toxicity of cyclophosphamide.

[0150] In this experiment, by Figure 4 As shown in Table 13, the number of mature follicles in the ovary was lower in the model group than in the normal group. The number of atretic follicles was higher in all groups than in the normal group. This may be because an increase in atretic follicles means a decrease in the available follicle pool (ovarian reserve). Even though the treatment group partially improved follicle survival rate, the overall number of follicles was still lower than in the normal group. The number of primordial follicles was slightly higher in the model group than in the normal group, and higher in the sample group than in the model group.

[0151] Table 13 Number of mature follicles in the ovary

[0152] control group Model group experimental group Number of mature follicles <![CDATA[2.00±2.00 a ]]> <![CDATA[1.00±1.73 a ]]> <![CDATA[0.67±1.15 a ]]> Number of atretic follicles 0.33±0.58a 2.67±2.08a 2.33±3.21a Number of primordial follicles 5.33±4.16a 5.67±0.58a 6.33±3.21a

[0153] (2) Observation of uterine tissue

[0154] Cyclophosphamide, as an alkylating agent chemotherapy drug, primarily exerts its toxic effects on the female reproductive system of rats on the ovaries. Therefore, the effects of cyclophosphamide on the uterus are mainly manifested in changes in the structure and function of the endometrium. The main changes include: ① Endometrial damage: Endometrial atrophy: Cyclophosphamide reduces serum estradiol (E2) levels, decreasing endometrial proliferation, leading to thinning of the endometrium, reduction of glands, and atrophic changes. Interstitial fibrosis: Long-term or high-dose treatment may induce uterine collagen deposition and fibrosis, affecting embryo implantation and pregnancy maintenance; ② Vascular and immune dysfunction: Disruption of vascular network: The drug inhibits angiogenic factors (such as VEGF), reducing uterine blood perfusion and further aggravating tissue damage. Local inflammatory response: Cyclophosphamide may activate macrophages in the uterus, release pro-inflammatory factors (such as TNF-α and IL-6), and exacerbate endometrial microenvironment disorder; ③ Association with ovarian function: Because cyclophosphamide directly damages ovarian follicles (such as depletion of primordial follicles and reduction of mature follicles), insufficient E2 secretion indirectly leads to loss of hormonal support for the uterus, manifested as the disappearance of cyclical changes in the endometrium related to the estrous cycle.

[0155] Depend on Figure 5 Analysis of uterine tissue revealed atrophic changes and collagen fibrosis in the model group. The experimental group, however, showed varying degrees of improvement in the endometrium.

[0156] 2.7 Tunnel Fluorescence Analysis of Histological Sections

[0157] Cyclophosphamide treatment significantly increased the TUNEL (terminal deoxynucleotidyl transferase dUTP-labeled) positive signal in female rat ovarian cells, indicating that it causes significant damage to ovarian tissue by inducing DNA breaks and apoptosis. In this experiment, the apoptosis-positive rate refers to the proportion of cells undergoing apoptosis in a specific sample, helping to study the effect of drug-induced apoptosis. (See Table 14 and...) Figure 6 As shown, TUNEL staining results (red fluorescence for apoptotic cells, blue fluorescence for nucleus localization, and only blue for normal cells) revealed significantly enhanced fluorescence intensity in the ovarian tissue of the Model group mice compared to the Control group, indicating that cyclophosphamide successfully induced ovarian cell apoptosis. The apoptosis rate in the sample group (6.19±2.97%) was significantly reduced to near the Control group level, with a significantly weakened fluorescence signal, suggesting that these two samples may have a significant protective effect against cyclophosphamide-induced premature ovarian failure by inhibiting the apoptosis pathway.

[0158] Table 14 Apoptosis Positive Rate

[0159] control group Model group experimental group Apoptotic cell positivity rate (%) <![CDATA[7.98±3.27 ab ]]> <![CDATA[12.2±2.1 a ]]> <![CDATA[6.19±2.97 b ]]>

[0160] 2.8 Ovarian stress markers

[0161] (1) GSH-Px level

[0162] GSH-Px is a key enzyme in the ovarian antioxidant defense system, relying on glutathione (GSH) to catalyze the breakdown of hydrogen peroxide (H2O2) and lipid peroxides, protecting oocytes and granulosa cells from oxidative damage. MDA is the end product of lipid peroxidation of polyunsaturated fatty acids (such as cell membrane phospholipids) under reactive oxygen species (ROS) attack. High concentrations of MDA can cross-link proteins and DNA, disrupting the membrane structure and function of oocytes and granulosa cells.

[0163] As shown in Table 15 of this study, compared with the control group, the GSH-Px content in the model group was significantly decreased (P<0.05), while the MDA level was significantly increased, indicating that the ovarian antioxidant defense system was damaged. Compared with the model group, the experimental group significantly increased the GSH-Px level in the ovary (P<0.05) and decreased the MDA level (P<0.05), indicating that sturgeon caviar peptides can improve oxidative stress-mediated follicular apoptosis and ovarian function decline.

[0164] Table 15 Ovarian Oxidative Stress Levels

[0165] control group Model group experimental group GSH-Px (ng / mL) <![CDATA[79.27±2.07 a ]]> <![CDATA[65.78±4.43 c ]]> <![CDATA[73.22±2.36 b ]]> MDA (ng / mL) <![CDATA[72.78±6.92 a ]]> <![CDATA[94.43±5.74 b ]]> <![CDATA[66.92±4.22 a ]]>

[0166] (2) Inflammation level

[0167] Cyclophosphamide treatment significantly increased the levels of interleukin-1β (IL-1β), IL-6, and TNF-α in the ovaries of female mice, indicating that the drug induced a local inflammatory response in the ovaries. This effect is closely related to oxidative stress and cell damage. As shown in Table 16, compared with the normal group, the levels of IL-1β, IL-6, and TNF-α in the model group were significantly increased (P<0.05), indicating that cyclophosphamide treatment increased the level of ovarian inflammation; compared with the model group, the experimental group significantly reduced the level of ovarian inflammation (P<0.05).

[0168] Table 16 Levels of inflammatory factors in ovarian tissue

[0169] control group Model group experimental group IL-1β (ng / mL) <![CDATA[56.52±4.57 a ]]> <![CDATA[89.22±6.81 b ]]> <![CDATA[21.26±1.81 c ]]> IL-6 (ng / mL) <![CDATA[320.00±10.6 a ]]> <![CDATA[376.40±36.65 b ]]> <![CDATA[336.40±15.81 a ]]> TNF-α (ng / mL) <![CDATA[40.69±4.88 a ]]> <![CDATA[356.80±19.61 b ]]> <![CDATA[124.60±8.68 c ]]>

[0170] 5. Conclusion

[0171] Cyclophosphamide damages ovarian function through a triple injury mechanism of oxidative stress, inflammation, and apoptosis, leading to follicle depletion and hormonal axis imbalance. Targeting these pathways (such as antioxidant, anti-inflammatory, or hormone precursor supplementation) can effectively delay premature ovarian failure, with the natural ingredient sturgeon caviar peptide showing significant potential.

[0172] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of sturgeon roe peptide in the preparation of a product for improving premature ovarian failure, characterized in that, The preparation method of the sturgeon roe peptide specifically comprises the following steps: (1) thawing the sturgeon roe, adding water, stirring uniformly, grinding and crushing, adjusting the pH value to 8 to obtain a sturgeon roe solution; (2) heating the sturgeon roe solution and then naturally cooling it; the temperature is raised to 80℃ and maintained for 10 min; and the temperature is naturally cooled to 55℃; (3) adding alkaline protease to the cooled sturgeon roe solution, stirring uniformly, performing enzymolysis, and then adding octenyl succinic anhydride sodium starch and vitamin E powder; the enzymolysis temperature is 55℃ and the time is 8 h; (4) enzyme inactivation, natural cooling, adjusting the pH value to 7 to obtain a sturgeon roe enzymolysis solution; the enzyme inactivation temperature is 90-100℃ and the time is 10-15 min; (5) performing coarse filtration and centrifugal treatment on the sturgeon roe enzymolysis solution, taking the supernatant, adsorbing through an active carbon series column, filtering, taking the filtrate, and obtaining a sturgeon roe peptide solution; (6) performing plate-frame filtration, taking the filtrate, high-temperature sterilization, vacuum freeze-drying, screening, and packaging to obtain the sturgeon roe peptide.

2. Use according to claim 1, characterized in that, In step (1), the mass ratio of the sturgeon roe to water is 1:6; the grinding equipment is a colloid mill and the number of times is 1; and the operation of adjusting the pH value specifically comprises the following steps: adding a saturated NaOH solution, stirring uniformly, and adjusting the material pH value to 8.

3. Use according to claim 1, characterized in that, In step (3), the addition amount of the alkaline protease is 1% of the mass of the sturgeon roe protein.

4. Use according to claim 1, characterized in that, In step (3), the addition amount of the octenyl succinic anhydride sodium starch is 10 g / L; and the content of vitamin E in the vitamin E powder is 50% and the addition amount is 12 mg / L.

5. The use according to claim 1, characterized in that, In step (4), the natural cooling is to room temperature; and the operation of adjusting the pH value specifically comprises the following steps: adding a hydrochloric acid solution, stirring uniformly, and adjusting the material pH value to 7.

6. Use according to claim 1, characterized in that, In step (5), the tool for coarse filtration is a filter bag and a 200-mesh screen; the centrifugal treatment speed is 5000-20000 r / min; and the number of times of adsorption through the active carbon series column is 3.

7. Use according to claim 1, characterized in that, In step (6), the filter paper precision for plate-frame filtration is 0.1 μm and the number of filtration times is 3; the vacuum degree for vacuum freeze-drying is 100 Pa, the temperature is -45℃, and the time is 2000 min; the screen mesh number for screening is 80 meshes; and the packaging material is a PE bag.

8. The use according to claim 1, characterized in that, The product is a medicine.

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